Illumina HiSeq 2500 sequencing; RNA-Seq of Group 2 innate lymphoid cells (ILC2) from uterine tissue (Virgin, E9.5, E18.5)
RNA-Seq of Group 2 innate lymphoid cells (ILC2) from uterine tissue (Virgin, E9.5, E18.5)
Single cell suspensions from Virgin and pregnant uteri were stained with CD127 (A7R34), ICOS (C398.4A), ST2 (RMST2-33), NK1.1 (PK136), CD3 (17A2), CD19 (6D5), GR-1 (RB6-8C5), TER-119 (TER-119), CD11c (HL3), CD8a (53-6.7), CD90.2 (53-2.1), CD45 (30-F11), CD11b (M1/70). All antibodies were purchased from Biolegend or BD bioscience. Viable cells were discriminated by using fixable viability dye ef450 from BD bioscience After sorting, uILC2 pellet were collected into fresh TRI Reagent (Sigma). RNA extraction was carried out by following TRI Reagent manufacture instructions. Transparent part containing mRNA was then purified with RNAeasy mini kit (Quiagen), gDNA contamination was eliminated with Turbo DNAse (Ambion) and we then clean up ILC2 mRNA with RNAeasy micro kit (Quiagen).  C57BL/6 (B6) WT mice were used at 8-12 weeks. Single cell suspensions from Virgin and pregnant uteri were stained with CD127 (A7R34), ICOS (C398.4A), ST2 (RMST2-33), NK1.1 (PK136), CD3 (17A2), CD19 (6D5), GR-1 (RB6-8C5), TER-119 (TER-119), CD11c (HL3), CD8a (53-6.7), CD90.2 (53-2.1), CD45 (30-F11), CD11b (M1/70). Viable cells were discriminated by using fixable viability dye ef450 from BD bioscience. Live, single cells, CD45+ve CD11b-ve NK1.1-ve, CD3-ve, CD19-ve, GR-1-ve, Ter119-ve, CD11c-ve, CD8a-ve, ST2+ve, CD90+ve were sorted with BD FACSAria III into PBS 2% FCS. After sorting, uILC2 pellet were collected into fresh TRI Reagent (Sigma). RNA extraction was carried out by following TRI Reagent manufacture instructions. Transparent part containing mRNA was then purified with RNAeasy mini kit (Quiagen), gDNA contamination was eliminated with Turbo DNAse (Ambion) and we then clean up ILC2 mRNA with RNAeasy micro kit (Quiagen).